Cody Sackett Christine Muñoz Juliet Kiyai-Bartlett
CHE 4080 Process Design II Spring 2016
May 6, 2016
Table of Contents
Executive Summary (Christine)………. 4
Scope of Work (Juliet)………. 6
Introduction (Christine)……… 10
Design of Base Case/Product Design (Cody)……… 12
Lab Work (Christine) ………... 36
Design Alternatives (Juliet)……… 39
Permitting and Environmental Concerns (Juliet)………...48
Safety and Risk Management (Cody)……… 51
Project Economics (Cody)………. 56
Global Impacts (Christine)……….. 66
Conclusions and Recommendations (Christine)……….. 68
Future Work (Christine)………... 69
Acknowledgements………71
References………..72
Table of Tables Table 1. Key Design Criteria ... 20
Table 2. BTEXterminator Raw Materials ... 26
Table 3. Simple Mass Balance ... 27
Table 4. Research/Lab Equipment ... 28
Table 5. Process Equipment... 29
Table 6. HAZOP ... 52
Table 7. Research Costs ... 56
Table 8. Process Equipment Costs ... 57
Table 9. Variable Costs ... 58
Table 10. Cash Flow Statement Template ... 59
Table 11. Economic Summary... 60
Table 12. Price summary ... 61
Table 13. Sensitivity Analysis ... 63
Table of Figures Figure 1. Strain Pathways ... 13
Figure 2. Aromatic Degradation ... 14
Figure 3. TOL & TOD Mechanism ... 16
Figure 4. PRB Type Curve... 19
Figure 5. Process & Production Diagram ... 21
Figure 6. BTEXterminator Production ... 22
Figure 7. Single Reactor PD ... 23
Figure 8. Parallel Reactors PD ... 24
Figure 9. Fibrous Reactor Scheme. ... 30
Figure 10. Media Test ... 37
Figure 11. MBR Module ... 40
Figure 12. MBR Demo ... 41
Figure 13. Hollow Fiber Reactor PD ... 42
Figure 14. Fibrous Reactor PD ... 43
Figure 15. Front Side Filtration ... 44
Figure 16. UV Filtration Pricing ... 46
Figure 17. UV Filtration Operating Costs... 46
Figure 18. Tornado Diagram... 64
Executive Summary (Christine)
BTEXterminator is a co-culture of two Pseudomonas putida strains that have been genetically modified to degrade benzene, toluene, ethylbenzene, and xylene (BTEX), as well as their respective isomers. Produced water is classified as any water that is returned to the surface due to the operations and production phases of oil wells, because it is preexisting in the
formation. This water is high in hydrocarbons, which includes BTEX, as well as, metal and salt.
Currently, there are few remediation processes available for produced water as it is normally injected into salt water disposal wells (SWD), or simply left in an open reservoir or tank where the BTEX will evaporate into the atmosphere before it is transported to disposal, both of which are costly and time consuming. Currently, there are no regulations on BTEX evaporating, and anticipated regulations on BTEX are the need for this project. Also, the option of an SWD is becoming less popular due to the increase in seismic activity near these wells. Produced water disposal is generally one of the highest lease operating expenses operators face. There are
opportunities to repurpose this water if it can be treated in a cost and energy efficient manner. As recently as 2009, it was anticipated that there are between 15 and 20 billion barrels of produced water generated in the United States annually.
Each individual P. putida strain naturally contains either the TOL or TOD pathway, which in combination, are responsible for the degradation of the components of BTEX. A plasmid is inserted into each strain in order to force both organisms to express both pathways actively. After successful modification is achieved, the co-culture will be established and cultivated up to scale via free cellular suspension. The product may then be transplanted into a reactor where it will be ready to treat produced water.
C&C has designed BTEXterminator with the potential to be as robust as possible; it can
handle a wide range of potential water volumes based on the clients’ demand, handle multiple
sets of water conditions, and the product has room for further adaptation as seen necessary.
However, this is not a system that can be applied to every situation in the field. BTEXterminator will only handle one step of the clients’ water remediation process, which is the removal of BTEX. Produced water will flow from a tank, reservoir, or well pad to C&C’s reactor(s). The water will then run through a various amount of reactors depending on the water composition and flowrate. BTEXterminator will remove all detectable amounts of BTEX so that the operator can handle the water as seen fit.
An economic analysis was done on a per skid basis to determine the feasibility of this
project. A per skid cost basis was generated with nine reactors assumed per skid. The IRR was
set to 40% to back calculate a yearly revenue of $6.12 million per skid in order to attain the
desired return rate. This project is not feasible when compared to the current prices of produced
water disposal. However, if the anticipated regulations are put into place, then this project
becomes increasingly favorable in the market.
Scope of Work (Juliet)
Design problem statement
Produced water makes up the biggest waste stream associated with conventional oil and gas production, as well as, unconventional petroleum resources. In a report drafted in 2011, it was estimated that roughly 7 to 10 barrels of water are produced for every barrel of crude oil
18. This produced water is usually considered a waste byproduct from oil and gas production. The oil and gas industry typically disposes of produced water by three main means: land application or discharge, subsurface injection, and offsite trucking. Land discharge is fairly inexpensive, but requires the water to be of higher quality. Produced water to be discharged to the surface must follow strict regulations set forth by Department of Environmental Quality (DEQ). These regulations ensure that the receiving waters and surrounding areas do not get contaminated. Re- injecting the produced water into the ground is a good alternative, but it can lead to an increase in earthquakes if the subsurface formation does not have the capacity to receive the water.
Therefore, the produced water can be trucked offsite to be re-injected into a formation that can accept it. Off-site trucking is very expensive for an operator
18. Since large volumes of produced water are generated during oil and gas production, it should be investigated for its potential use as an alternative water source.
The characteristics of the produced water directly corresponds to the geographical location from which it originated. Furthermore, the quality of the produced water is affected by the type of hydrocarbon produced, as well as, the geochemistry of the producing formation. An analysis of produced water will find both inorganic and organic constituents. The most commonly found organic constituents in produced water are benzene, toluene, ethylbenzene and xylene
compounds (BTEX). C&C can create its own niche in the water treatment industry by focusing on removing certain contaminants in the produced water, specifically the BTEX compounds.
BTEXterminator is a co-culture of two Pseudomonas putida strains that have been genetically
altered to completely degrade BTEX and its respective isomers. Removing BTEX could be the
first step necessary in converting the produced water from a waste product into a viable alternative water source for numerous applications.
Constraints on the design of BTEXterminator
In the early design of BTEXterminator, C&C considered the following constraints:
Economic
o C&C will only remove the BTEX compounds in produced water. Therefore, the entire process needs to be as inexpensive as possible since the water will require further treatment in order to be considered an alternative water source. If the price per barrel of treated water is much lower for C&C's process, it would attract customers and make this an economically viable project.
Environmental
o The water exiting C&C’s process needs to meet the state regulations on BTEX levels which varies depending on how the water will be used. C&C will focus on meeting the state regulations for BTEX concentrations in drinking water. Since we are only focusing on removing BTEX from the produced water, the water will need further treatment to be safe for drinking.
o C&C will need to consider the potential for genetic transfer between the genetically modified P. putida strains and any microorganisms found in the produced water. The reactor residence time is one hour, so any genetic transfer that could occur should be very slow. If C&C finds that the microorganisms found in the produced water can be altered by the P. putida strains, then a front-side ultrafiltration membrane and UV disinfection system would be added to the process. This front-side ultrafiltration membrane and UV disinfection system will prevent any microorganisms in the produced water from reaching the reactor housing the co-culture of P. putida. The back-side ultrafiltration membrane and UV disinfection system will ensure that the genetically modified microorganisms do not leave the process or enter the environment.
Sustainability
o The goal of BTEXterminator is to remove all BTEX compounds in the produced water fed to the reactor. Although the co-culture of P. putida uses BTEX as a carbon source, it will need other nutrients to perform efficiently. There is a basal mineral salts medium that C&C will use during the research phase, but an alternative nutrient will need to be investigated during scale-up.
o The two P. putida strains aerobically degrade BTEX, therefore, C&C will need to look into a safe oxygen source for the organisms on the skid. The current design uses hydrogen peroxide as the oxygen source for the microorganisms based on lab-scale studies done by Shim
11and his colleagues.
o C&C will need to figure out how long the P. putida strains can live in the harsh environments of produced water. P. putida was found living in an environment with high metal content; it also has a certain level of halotolerance.
Manufacturability
o C&C plans to be able to move all the equipment necessary to treat the produced water on a skid. Therefore, all the process equipment needs to be reasonably sized to allow for easy transportation with minimal costs.
The preliminary design shows that a skid-able 5000L reactor will work.
o The materials chosen for the process equipment should minimize capital costs as well as costs associated with maintenance.
Ethics
o The use of genetically modified organisms is a very controversial topic in this country. As a result, C&C will need to take extra precautions to make sure the genetically modified P. putida strains are not released in the treated produced water. The co-culture of P. putida will be immobilized on the fibrous bed reactor so that a high percent of the cells remain in the reactor. The ultrafiltration
membrane and UV disinfection at the end of the process will prevent the release
of any genetically modified P. putida strains. Educating industry and community
members about the safety of our product will be important when implementation of the project begins.
Health and Safety
o BTEXterminator needs to remove all detectable BTEX compounds in the produced water.
o Transporting oxygen safely unless a different oxygen source is found.
Introduction (Christine)
The operation of oil and gas companies has affected not only the world economy, but also the world’s water supply. This significant effect of the petroleum industry is felt all around the world. As recently as 2009, it was anticipated that there are between 15 and 20 billion barrels of produced water generated in the United States annually.
12Produced water is water that has been returned to surface because of drilling, completing, or producing a well. This is not water that has been injected into the reservoir, but water that was already existing in the earth. The composition of produced water is difficult to quantify, however, there are usually salts, metals, hydrocarbons, and reservoir bacteria present. Some of these hydrocarbons are harmful and there are not currently specific regulations on the components of BTEX (benzene, toluene,
ethylbenzene, and xylene). That is, there are not regulations deterring operators from disposing of produced water that contain high levels of BTEX.
In the United States, it is up to the state government to set and adhere to regulations on the acceptable levels of BTEX in waters. The states of Colorado and Wyoming classify the acceptable levels of BTEX based on the use of water, and as a result the regulations for both states are very similar. C&C has set out to provide an organism capable of efficiently removing BTEX from this water so that it may cheaply be treated further to meet different specifications depending on desired use of the water.
The cost to dispose of produced water is very high for operators. In fact, it is about a third of the leasing costs associated with operations. There are anticipated regulations on the disposal of BTEX in produced water, which would make BTEXterminator a competitive product. C&C sees a lack of this technology in the petroleum industry, and the potential of BTEXterminator in that market share.
C&C has created a new product, BTEXterminator, to meet this need. Many bacteria are
naturally able to degrade one or many of the components of BTEX. C&C has investigated
Psuedomonas putida, which is the bacterium used for BTEXterminator. This bacterium was
chosen because it has been isolated from the discharge of a petroleum refinery. While other
bacteria could be used in BTEXterminator, C&C is confident that P. putida is the best choice.
Through genetic modification, BTEXterminator is an effective method of removing BTEX in large volumes of produced water. BTEXterminator is a co-culture, meaning that there are two strains of P. putida in the product. An advantage of using a co-culture is that one strain has the natural capacity of degrading about half of the components and the other strain has the capacity to degrade the other half. Such complementary strains are BTE1 and TX1.
C&C will attempt to make BTEXterminator as robust as possible armed with the
information gleaned from the formations in the region. Genetic modification will be necessary in order for the organism to effectively degrade all components of BTEX, and survive in the harsh conditions of produced water. Hydrocarbons need to be the first components removed from the water in order to undergo further purification processes because of the damaging effects of hydrocarbons on this equipment.
Once the produced water has had BTEX removed there are many possibilities for further treatment of the water in order to reach a specific purity specification. C&C has determined that the best business strategy is to make the microorganism capable of removing BTEX completely, so that it is undetectable. Regardless of the consumers’ use of the water they can undergo any further processing to meet their specific needs. Environmental discharge is a possibility if the salt concentration is reduced to ground water regulations. Since Wyoming and Colorado are agrarian states, the water could be used for agriculture, which requires large quantities of water. The choice of the end use of the water is in the hands of the client.
Currently, there are some ways to bioremediate produced water, but there is not justification for the current open system processes due to the lack of regulations. Thus,
BTEXterminator is a revolutionary product in a market that is anticipated to arise. C&C realizes that once there are regulations, operators will need this product to meet the required
specifications of produced water before disposal. So, the current options of salt water disposal
wells or leaving the water in an earthen pit or tank will not be available until the water is rid of
BTEX.
Design of Base Case/Product Design (Cody)
Project Definition
C&C has created a new product, BTEXterminator, which can meet the need of ridding produced water of BTEX. The composition of produced water is difficult to quantify; however, it is known that high concentrations of salts and some heavy metals can be present. Also, there are other hydrocarbons in the water, which BTEXterminator is also capable of consuming. This information can be determined from studying the formations in which operators will be fracking.
Once the produced water is characterized by region and formation, the composition will be predictable. Genetic modification will be necessary in order for the organism to effectively
degrade all components of BTEX, and survive in the harsh conditions of produced water. C&C is focused on only removing BTEX from produced water. As a result of our product being
specialized to this task, we really only have two process sections that we need to focus on, one of them being the production and design of BTEXterminator. The second section is the ability for BTEXterminator to perform within a reactor and remove BTEX in an industry setting.
Product Description
There are many bacteria that naturally are able to degrade one or many of the components of BTEX. In order to capitalize on this natural ability, C&C has investigated Pseudomonas putida and plans to use its natural abilities through genetic modification in order to produce an
effective method of removing BTEX in large volumes of produced water. Some of the strains of this bacterium are naturally capable of using pathways for the degradation of components of BTEX, and thus give a great starting point. BTEXterminator is a co-culture, meaning that there are two strains of P. putida in the product. There are many advantages of using a co-culture. The main advantage is that strain A has the natural capacity of degrading about half of the
components and strain B has the capacity to degrade the other half. Since these strains are
complementary, it is very beneficial. In literature, it was found that the two strains that
complement each other in this way were BTE1 and TX1.
BTEXterminator will start out as two separate strains of Pseudomonas Putida, TX1 and BTE1. Each strain naturally contains one of the pathways necessary to degrade particular
components of BTEX within produced water. Natural expression of this pathway means that it is a chromosomally expressed trait. TX1 contains the TOL pathway, which makes it possible for the strain to consume toluene and xylene, as well as all possible isomers. BTE1 contains the TOD pathway, which is complementary to the TOL pathway by giving this strain the capability to consume toluene, benzene, ethylbenzene, and all of their respective isomers. Figure 1 can be seen below which gives a more organized representation of each organism’s ability to degrade BTEX
6.
Figure 1. Strain Pathways: It is worth noting that o-xylene is not k nown to be consumed by either pathway but has such a low selectivity that it is usually not present in detecta ble amounts, as is true with o-toluate. 6
(Christine Chemistry Section)
The degradation of aromatic compounds is very complicated, as Figure 2 shows.
However, the pathways can be simplified because each component of BTEX is funneled into the
same intermediates and eventually are degraded by the same pathways. However, the way the
ring is broken and cleaved is specific to either the TOL or TOD pathway.
Figure 2. Aromatic Degradation: Common pathway of aromatic degradation in bacteria, large and difficult to see it manages to visualize to the reader the complexity of the biological pathway.30
The degradation of BTEX is done aerobically. The easiest component of BTEX to degrade is toluene. In order to facilitate degradation dissolved oxygen must be present to be used for ring activation and cleavage. Also, oxygen is the electron acceptor to allow for complete degradation. The TOL pathway is facilitated by an enzyme, monooxygenase, and the TOD pathway uses the enzyme dioxygenase. The TOL pathway attacks the constituents of an aromatic ring, such as the methyl and ethyl. These substituents are transformed by oxidations; methyl to pyrocatechol and ethyl to phenyl glyoxal. The TOD pathway attacks the aromatic ring by forming 2-hydroxy-substitued compounds. The benzene oxidation is catalyzed by the dioxygenase and is a hydroxylation. Both of these pathways converge in the formation of catechol and its intermediates. The catechol intermediates are then degraded into compounds such as pyruvate and acetaldehyde. These metabolites can then go into the Krebs’ Cycle,
eventually flowing into the Electron Transport Chain and creating energy for the cell. The Krebs’
Cycle, Electron Transport Chain, and energy creation are a part of aerobic respiration that is done in all aerobic bacteria.
Figure 3 shows the simplified version of the pathways. The dotted lines represent the
TOD pathway and the solid lines represent the TOL pathway. As Figure 3 shows, each of the
pathways funnel into the TCA cycle.
Figure 3. TOL & TOD Mechanism.29
(End Christine Chemistry Section)
Genetic modification will then be undergone by each strain separately in order to ensure proper modification. Whichever pathway the strain does not have naturally available to it will be inserted into the organism via plasmid genetic modification. Since both our organisms only differ by strains, and not family or species, they both will require similar conditions to uptake a TOL or TOD plasmid. The TOL plasmid was first characterized in Pseudomonas aeruginosa in 1978, and was found to be highly thermo sensitive. It was found to become unstable at 42
0C
7. In a similar study, the mechanism of the TOL plasmid uptake was explained. This plasmid was introduced to a strain of Pseudomonas putida at 30
0C
8. In both of these studies, the pressure was not indicated, so it is assumed that atmospheric pressure was the condition at which the strains of bacteria were modified. The TOD plasmid is very similar to the TOL plasmid as it is comprised of the same types of pathways, but degrades different components of BTEX than the TOL pathway. Thus, it can be assumed that the TOD plasmid uptake is similar to that of the TOL plasmid.
After modification is complete, each strain will be able to express one of the pathways on
a chromosomal level and the complementary pathway will be expressed via plasmid expression.
This dual expression will be beneficial to the organisms in co-culture because regardless of any lateral gene transfer (gene transfer amongst members of the same culture) we have ensured that BTEXterminator will always have both pathways expressed chromosomally and plasmoidally.
Vertical gene transfer (gene transfer to offspring) will occur as well as part of the organism’s life cycle and both forms of expression should be retained in the long term. This means once we have modified one culture we should simply be able to grow that culture up into quantities necessary to provide our clients with the proper amounts of BTEXterminator and will not need to go back to lab scale operations and undergo the genetic modification again with the original strains.
C&C will then need to cultivate the strains up to larger and larger scales until ready to be introduced to a reactor environment and one another. Pure strains post modification will be streaked and kept in deep-freeze as a reserve. The organism may be modified further after initial trials and research has begun, so it would be beneficial to have each version of our organisms available in reserves. These two separately modified strains will then be introduced to each other in the same environment, again only differing by strains and not species is incredibly
opportunistic for us because both organisms require the same nutrients and minerals necessary for their survival, these will be listed later in the material balances section. Equal amounts of each strain will be introduced in the same tank, which will be C&C’s stock and reserve for BTEXterminator. The size of this tank or tanks will depend on the companies stage in its timeline, but it will be large enough to supply clients as well as maintain an active and ready to go reserve. This ends the production of BTEXterminator, the lab scale operations should only be done with an incredibly small amount of the organisms (aside from research), and then they should be self sustaining with the right media to grow and cultivate on their own to the proper portions for C&C’s client demand.
General Process Description
Once BTEXterminator has been produced, it completely depends on the client’s needs
how much of the product they will need. C&C has made the process as robust as possible with
the hopes of meeting as many situations as possible. The process design is really to test the
feasibility of the product; it was the only way C&C could rationalize evaluating the progress made but is not the scope of this project. The organism will be able to survive in a wide range of salt and metal concentrations, as well as, BTEX. Reactor scheme will vary greatly depending on the amount of BTEX and the amount of water a client wants processed, this will change the number of reactors, and perhaps even the number of passes necessary. Design of the reactor will not vary however, and C&C plans on sizing a single type of reactor and making the technology skid-able. So rather than change the reactor size, the amount of BTEXterminator in a reactor, or even residence time of water in the reactor, we will be simply adding more reactors or more passes to meet demand. C&C will be using a fibrous bioreactor in which BTEXterminator is immobilized on the fibrous material; this will make the remediation process more efficient as well as help ensure that BTEXterminator does not get flushed down stream.
With that being said, C&C envisions being hired by a client with a specific desired flowrate for treated water and it will be C&C’s job to utilize BTEXterminator to deliver the client that flowrate of BTEX free water. Produced water is returned to the surface in very large volumes, we have found estimates from industry sources such as, Don Whisonant who is an Engineering Advisor for Devon Energy, that tell us over a single oil well’s lifetime, it may produce anywhere from 100k to 2000k barrels of water
9. Clients will very likely have several wells close to each other and it is unlikely they would have a need for such a high volume of water instantly. Thus, C&C anticipates that our clients will store this water in a reservoir; they may then send their desired flowrate of produced water to BTEXterminator to treat the water and meet their water demand. If storage is not an option adding/removing reactors to handle a
varying flowrate is no issue as long as the water is characterized.
From a reservoir or formation, produced water will be pumped to C&C’s reactor scheme
at a constant flowrate. The flowrate from a single well will vary and constantly decrease over its
life, however, water will be delivered from multiple wells. C&C will also only run water for the
first three months of the well’s life; typically, 90% of produced water is forced to surface in the
first three months
9. It would be more economically realistic for the operator to ship the small
amount of water, the other 10% over the rest of its life. These wells can have a production
lifetime of a few years up to periods of 30 or 60 years, it really varies, but the first three months always produces a majority of the water. As seen in Figure 4, the majority of the produced water is forced to surface in the first three months.
Figure 4. PRB Type Curve. Type curve of a typical well in the Powder River Basin.9
Enough reactors and BTEXterminator will be provided in order to ensure complete degradation of BTEX to untraceable amounts, these reactors in parallel will be scaled back as water production decreases over those three months. The water will then exit the reactor scheme and run through a filtration system so no GMO is released downstream. This BTEX free water will then be turned over to the client and be treated further depending on their desired use for the repurposed water. This may include environmental discharge, agricultural use, or even drinking quality.
Base Case Design Assumptions
In order to find an appropriate estimated size for our reactor and the amount of
BTEXterminator to treat a certain amount of water, C&C ran some very rough estimates. These estimates are based off of a target 5000 bbl/day of produced water treatment. This volume would treat two sections typical of the Powder River Basin according to Don Whisonant
9. Sections in the oil and gas industry are defined as a 1 square-mile area. Typically, in the Powder River Basin
0 100 200 300 400 500 600 700 800
0 50000 100000 150000 200000 250000 300000 350000
1 32 60 91 121 152 182 213
Powder River Basin Parkman 9500' Lateral Water Production Profile
Cumm Water stb/d XR Average Water stb/d
Cummulative Water
Month of Production
Water Production
there are two well pads that each run to two wells within that square-mile. A series of backwards calculations were done based off of approximated literature results. C&C took an ideal target degradation rate we would like to achieve in BTEXterminator , and tried to match the cell concentration necessary to acquire that rate of degradation in our reactors. Based off numbers from Devon Energy, C&C sized the base case to treat 25,000 L/hr, a decent expectation for that square-mile area. The concentration of BTEX within that water was assumed to be constant.
These calculations led to an estimated reactor size, BTEXterminator per reactor, and a single reactors production ability per day as displayed in Table 1
10. It was then clear that multiple reactors would be necessary for the base case scenario.
Table 1. Key Design Criteria: Single reactor size and production ability.
Process Diagrams
Figure 5 displays the big picture of C&C’s envisioned role for BTEXterminator. It shows
unaltered produced water flowing into our remediation process. Our product then treats the water in order to remove BTEX where the water is then returned to the hands of the client. C&C has split its responsibilities up into two key sections, the modification and cultivation of
BTEXterminator, and the products ability to perform within a reactor and actually remove
BTEX, both of which can be seen below.
C&C will be focusing on these two sections of produced water
treatment
Filtration
Figure 5. Process & Production Diagram: The overall process and production diagram shows BTEXterminator’s role in the water treatment process.
The first step is for BTEXterminator to undergo its genetic modification and be cultivated to a point where introduction to the reactor is appropriate. Figure 6 displays the process that each individual strain of P. putida goes through before they are introduced as a co-culture and
transplanted to the reactor shortly thereafter.
After BTEXterminator is produced and cultivated to scale within a reactor, it becomes the key part of the BTEX removal process of produced water. Figure 7 displays this process
flowsheet for a single reactor being fed produced water from a reservoir at a constant flowrate.
The additional nutrients and supplements feed consists of the basal mineral salts and the oxygen source. This process is as simple as depicted in this process diagram; the real design work is put into BTEXterminator. C&C has designed a single reactor size as well as estimated amount of organism necessary to treat an estimated BTEX concentration. Based off these assumptions and those made previously, a single reactor could treat approximately 570 bbl/day of produced water
Figure 6. BTEXterminator Production: This figure expands on the GMO production section C&C will be focusing on, displaying a hierarchical order each strain will go through to create BTEXterminator.
for BTEX if operated over the 24hr period. Though this is the max treatment rate for a single reactor, as long as BTEXterminator is provided enough media, oxygen, and BTEX to survive the reactor does not have to be at full capacity. C&C have also anticipated a skid-able design where these reactors could simply be placed in a parallel scheme to handle more, or less water as shown in Figure 8.
Figure 7. Single Reactor PD: The bioreactor where BTEXterminator is housed is shown being fed a constant amount of produced water from a reservoir.
Media/H20 2
Figure 8. Parallel Reactors PD: Proposal for reactor scheme, each reactor would be the same size and treat the same amount of produced water . The number of reactors would be scaled with the desired production. A membrane and UV filtration would lik ely be the last step in order to inhibit any GMO or biomass from moving downstream.
M edia/H202
After the water had left the reactor system it would be sent through a membrane filtration unit. A membrane should be capable of stopping any GMO from moving through but still allow for the water to flow with little to no restriction. Since public perception of GMO’s is so
negative, along with the unknown consequences of releasing BTEXterminator into the
environment, C&C will also include a UV filtration step to kill any remaining organisms prior to the water being released to further downstream operations. It is important to keep in mind this system represents the concept of the design BTEXterminator will work in, but will include 9 not 5 reactors, and will include the filtration equipment.
Material Balances
Creating BTEXterminator is all done on small lab scale operations; the genetic
modification is done on this scale so that we ensure the colonies that are selected uptake the
plasmids, and that we do not have separate colonies, one of which would be resistant to the
transformation likely. To successfully do this in lab a material balance is done around the
production of BTEXterminator shown in Table 2. The components listed in the media would
simply be scaled up along with the organism’s volume. TX1 & BTE1, along with the plasmids
used in the modification, would be used in an incredibly small amount as shown, again to ensure
plasmid uptake and expression.
This provides C&C the ability to scale the required materials for
BTEXterminator’s survival so that it may operate at optimal conditions. Since C&C also is focused on the performance within produced water a simple mass balance was done around the inputs and outputs of the reactors. Table 3 displays a simple mass balance, as well as a list of additional key components within produced water that are of importance to our organism’s abilities to perform.
Table 2. BTEXterminator Raw Materials: A materials list/balance that represents inputs required for laboratory operations which can be scaled up to larger cultures.
Although very simplistic this mass balance serves its purpose when trying to understand our products aside from water, by only removing BTEX the only byproducts we would get would be as a result of the organism’s metabolism, even with significant literature it is difficult to anticipate exactly what biomass includes. BTEX may also not be completely removed, and likely would not be, but BTEXterminator’s purpose is to remove detectable limits so complete degradation was assumed for this balance. Additionally, salt and metal content ranges were established from the US Department of Energy, these were then compared to the produced water sample we based our calculations off of which were provided by research done at the University of Wyoming
11, 12. Without lab research it will be impossible to really utilize these numbers and understand if our product will be comfortable with our concentrations, let alone the entire ranges of those concentrations.
Equipment Lists
C&C is also able to split up our equipment lists based off the section we look at, whether GMO production or water treatment sections. For the production of BTEXterminator some specific lab equipment will be needed in order to run experiments and collect answers necessary to scale up to large operations, or even simply create the product. A simplified list can be seen in Table 4.
Table 3. Simple Mass Balance: A simple mass balance around the reactors is done with concentrations included for other components of interest that will not be consumed.
This lab equipment would be a great start to producing a sort of prototype of BTEXterminator, which then could be researched to progress closer to becoming a viable industry ready product. A separate equipment list is seen in Table 5 where the equipment that is anticipated to be required to actually treat the water is listed.
Table 4. Research/Lab Equipment: A preliminary list of equipment necessary for accomplishing the production of BTEXterminator.
These two sets of equipment lists are preliminary by the fact they are merely anticipations, some categories listed may include more than one component or piece of equipment but these lists will be used for a preliminary economic analysis.
Reactor Design
Only a single type of reactor will be designed and used for BTEXterminator to perform.
It will be modeled after the design that uses a fibrous bioreactor, which C&C found to be the most promising
10. This reactor will be suitable as a healthy environment for BTEXterminator to thrive and remove BTEX efficiently. The reactor layout is simply a 5000 L (app. 32 bbl) tank lined and coiled with a fibrous material, which will serve as a means to immobilize
BTEXterminator. It is assumed the cotton innards can operate well over a year before too much wear and tear takes plus, but other materials such as nylon or industrial carpet may be
considered. Immobilizing the organism ensures that the fibers will be saturated with the product throughout the reactor, and the rate of BTEX degradation in the reactor will not vary based on location within the reactor. Figure 9 is a very simple layout of the reactor construction.
Table 5. Process Equipment: Anticipated equipment requirements to treat produced water with BTEXterminator are listed.
Through experimentation, the optimal temperature for toluene degradation was found to be 35
0C. However, the optimal temperature for degradation of both toluene and benzene was found to be in the range of 15-35
0C. The optimum temperature to degrade all components was found to be 33
0C
13. The optimum pH was found to be within the range of 6-8, which is not surprising. Specifically, degradation rates increased the most in the range of 7.5-8. These measurements are assumed to be taken at atmospheric pressure. Produced water is not typically outside of this pH range, unless large amounts of acid producing bacteria or acid formations are being dealt with. This is not common in the Powder River Basin, treating any large pH change with this much water would be impractical for BTEXterminator at this time so the pH
monitoring is really to watch organism viability. If it changes too much then correcting 25,000 L/hr would not be ideal, other routes would need to be considered at this time (unless
BTEXterminator is modified to handle a wider pH range).
Figure 9. Fibrous Reactor Scheme.6
This reactor serves only as a housing unit for BTEXterminator and does not have operating specifications outside the conditions necessary for BTEXterminator to survive and perform. However, there are a few important design criteria that we hope to match on a large scale reactor and not just a lab scale. We expect this design to lead to greater than 90% of the cellular mass being immobilized within the reactor. If this is accomplished, then it would be expected to have a very high cell viability. Lab scale studies suggest that the reactor should be able to perform well over a year if these conditions are met. This is a huge estimation and would likely change under produced water conditions and scale up. Upon start-up, C&C would likely resupply BTEXterminator, as well as, the fibrous material within a reactor every three months, then slowly increase turnaround frequency as the system became better understood.
Further Considerations for Reactor Design
Though the reactor design is fairly simplistic the fact that it is housing a biological environment produces many challenges that need to be addressed. There are three immediately noticeable conditions that are detrimental to our system; temperature, pH, and oxygen
availability. The optimal ranges and targets for these conditions have been discussed but the solutions to maintaining them have not. Materials necessary for the design are a key design feature as well. C&C needs to minimize the cost while ensuring the material is appropriate for the organism’s survival, and does not allow the release of BTEX into the atmosphere via diffusion through improper materials.
Material selection will be the first area of interest discussed. In the original design of this
reactor on a lab scale operation a glass tank was used in conjunction with stainless steel and
cotton serving as the fibrous material. While this is fine for lab scale a glass reactor on site is not
an option and the stainless steel assumption made last semester is simply more expensive than
the materials need to be. Rather C&C will be using fiberglass as the material of choice for both
the construction of the reactor tank and the infrastructure to support the fibrous cotton innards.
This will not only serve appropriately as a material choice, but it will reduce the capital costs of building a reactor as well as the reactor weight. The main concern for moving away from
stainless steel was ensuring that the BTEX components would not diffuse through the system and into the surrounding atmosphere, defeating the purpose of BTEXterminator. With only a one- hour residence time in the reactor, C&C is confident that none of the constituents will be able to diffuse through a fiberglass shell. This can be verified very easily during laboratory testing before any pilot scale up process is considered.
Temperature is one of the most important operating conditions that needs to be met in order for BTEXterminator to degrade efficiently. Being a biological system the temperature range is very small, either meet that range, or fail to degrade BTEX and possibly kill the organisms. C&C has identified two possible ways that the temperature in the reactor could deviate from our range; ambient influence and feed temperature. Since our reactor is a skid design the system will be operating in the oil field directly. Ambient temperature will vary greatly depending on region and season but it is easy to see how running in the subzero temperatures of Wyoming could cause a problem. Now not all reactors will need to have a temperature control system if they are not operating in these widely varying ambient temperature areas but the possibility still needs addressed. Another possibility is for the temperature of the produced water in the feed to the reactor being drastically different from what the organism would like. The temperature of produced water directly from the formation will likely vary greatly depending on formation type. Unfortunately, there is not a lot of readily available information on the temperature of produced water so C&C is required to speculate at this time.
The water could be as cool as the ambient temperature at the time or nearly as hot as the ambient temperature; so 5⁰ C up to 75⁰ C would not be unreasonable. C&C’s solution to these
temperature concerns is to utilize our short one-hour residence time. The issue of ambient
temperature would be an easy design fix, simply adding insulation to the system in combination
with the one-hour residence time would be enough to negate ambient temperature concerns. In
theory our ideal operating temperature is 33°C, we do not know how quickly efficiency and viability decrease as that temperature is deviated from so specifics on the insulation and heating needed for the system cannot be determined, but instead discussed. In terms of insulation there are single layer and multilayer configurations available, it varies depending on the material selected along with the size of the bioreactor
8. Insulation need will also change depending on the rest of the temperature control system; this could include the water coming in heated or cooled, constant heat being delivered to the bioreactor to maintain a temperature range, or a combination of both. Either way a cost analysis would be done; this would determine whether less efficient insulation and more utilities, or expensive insulation and lesser utilities is more cost effective.
Heating the feed water out of formation would be the easiest direct solution to temperature concerns; simply adding a natural gas combustion heater prior to the water being sent to the reactor would do the trick, and one would think utilities would be reasonable since natural gas would likely be available on site (if not consider propane or diesel). Cooling the water, however, would be a large challenge. It would take time to cool the water 20⁰ C-40⁰ C without a large refrigeration system which would mean the process could not be done immediately and the water would pile up and sit. It is not economical to cool such large amounts of water at this time; this system may not work for all produced water scenarios. At this time C&C would probably only be able to handle water at or cooler than our desired range; the cost, size, and time of a system to cool water down to our range would be far too much for a skid system. A central remediation facility where the water would be shipped too would be a better option at that time. If it is determined in lab our organisms need a very small defined temperature range to operate then the cost of the solution to our temperature control issue becomes much greater, this costs would be assumptions on assumptions at this point in the design, and will not be accounted for in detail.
Instead, when determining the cost C&C would charge to the operator a 40% increased IRR will
be used to account for risks and uncertainties such as these.
Another important operating condition for a biological system to maintain is the pH. For our system to run efficiently the organisms need to remain healthy and viable, too acidic or basic of conditions would lead to the death of our product and the inability to remove BTEX. With a typical pH range of 6.5-8 produced water is not too extreme in this category
8. It is likely that BTEXterminator would be able to operate within this entire range of pH values, it just may be more efficient closer to a neutral 7. If the pH happened to fall outside of that range C&C would recommend shipping the water to a fixed facility where batches of produced water could have pH corrected before potentially harming BTEXterminator. Attempting to change pH by even a little bit with such large amounts of water on site, where the operation would like to be as close to continuous as possible would be infeasible. During startup years, which is all that is accounted for in this analysis, building a skid/system with a pH correction system that would need to be used less than 1% of future scenarios would not be advisable. Maybe as the company scaled up, and if pH issues were ever actually an issue, C&C could incorporate a larger pH range into the organism or add a correction system.
Lastly, another large concern of C&C is oxygen availability to our organisms. These organisms operate under aerobic conditions meaning they need oxygen available to them as an electron acceptor in order to perform many of their biological metabolic pathways. In aerobic systems aeration is performed in order to continuously supply the system with oxygen. Normally atmospheric air is introduced into the system and circulated. The purpose of BTEXterminator is to degrade BTEX without releasing any of it into the atmosphere. If air were to be bubbled into the system it would cause an increase in pressure and nitrogen buildup that would need to be properly vented, to avoid pressure buildup. This could be done if the vent contained a filtration system for BTEX. Filters for constituents such as benzene do exist and are used in large scale industrial applications and even individual personal pieces of protective equipment, one that is commonly used on small individual gas masks is an activated carbon and potassium
permanganate filter which could easily be fitted to one of C&C’s bioreactors. Another route to
completely maintain a closed system, no ventilation or anything like that, would be to use a chemical additive as an oxygen source. Hydrogen peroxide is commonly used in lab scale techniques but would likely be too pricey for our final skid design. Most other chemical that could be selected would also be considered additional pollutants, overall C&C will still use hydrogen peroxide at this time but will continue to look into optimizing this cost.
Base Case Assumptions
Below is a list of the many assumptions made throughout the design of the base case.
Base Case Assumptions
o Target Degradation of 600 gm/L-hr at the concentration of 15.4 gm/L o 25,000 L/hr produced water flowrate to base case
o 3 Month operation (90% of the well’s lifetime water achieved)
o Produced water pH is not found outside the operable range of our organisms o Hydrogen Peroxide would serve as an acceptable oxygen source
o Cotton innards operable for 4 jobs without replacement, consider nylon
o 1-hour residence time allows for complete degradation (Below Detectable Limits) o Fibrous scheme, membranes, and UV filtration completely reduce risk of GMO
release downstream
o Single reactor can treat a maximum of 570 bbl/day as design but may treat lower levels if nutrients/supplements are still fed (reactor does not have to be full, handles lower rates)
o Fiberglass reactor materials in combination with 1-hour residence time will not allow volatile hydrocarbons to diffuse out of the reactor and into the atmosphere o Present design may not be a one size fits all solution
Large temperature corrections may not be possible and minor ones are
assumed to be included with our high risk 40% IRR
Lab Work (Christine)
C&C had access to the specific strains of P. putida discussed in the literature.
Characterizing the viability of the organisms in produced water is paramount to the
understanding of how effective BTEXterminator can be. There are many tests to be completed, but in the time restraint, it is unrealistic to complete all of these experiments. Thus, only a few of the experiments were chosen to complete.
In order to complete experiments in the lab, employees must go through specific training.
These were offered through the University of Wyoming Environmental and Health Safety Department. The class names were Chemical Hygiene, Hazardous Waste Generator, and Chemical Safety Handling-Benzene. These classes trained employees to work safely and effectively in the laboratory environment. Once these classes were taken, employees were then able to work in the lab.
Before experimentation could be completed, the bacteria needed to be grown using rich media. Specifically, Lysogeny broth was used for this purpose. The media is sold in powder form, which can then be mixed with water to make either liquid media or media with agar. Both of these options were chosen because the different experiments needed either both or either the liquid or solid media. Once this media was mixed, it was then autoclaved to achieve
sterility. Figure 10 shows what was done in the lab this semester.
Figure 10. Media Test: On the left, inoculated media with P. putida. On the right, sterile media for comparison.
Sterile technique was used in all of the experiments. Then, the liquid media was inoculated with either BTE1 or TX1 to be grown for approximately forty-eight hours. This inoculated liquid media can be used to make reserve stocks of the bacteria in case of contamination or the need for more bacteria during experimentation. Each strain was also streaked onto plates so that individual colonies could be identified. This is important for several reasons. Namely, possible contamination can be visually spotted on the plates and single
colonies can be used for further experimentation.
The other experiments to be completed are testing salt tolerance and understanding how
the bacteria grow on a carbon source that is a component of BTEX. In order to test the salt
tolerance, a carbon source of benzoate will be used. This makes it so that the experiments don't
have to be done in the hood, and can instead be done on the bench table. Since the bacteria is
also to be tested with a carbon source of BTEX, the understanding of how the bacteria behave with that carbon source will be known.
In the future, the genetic modification of each strain of P. putida must be done. There are many ways to force the uptake of a plasmid in bacteria, and the method depends on the
bacterium. Typically, Pseudomondas are not difficult to modify. Typical research into modifying P. putida with the TOL and TOD vectors involves the simple transfer of vectors through
conjugation. This means that the vectors are transferred through contact of the donor and recipient cells.
31To measure the degradation rate of the components of BTEX from each strain and the co- culture, media containing all necessary nutrients except carbon would be inoculated with the strain of P. putida. Then, a component of BTEX in vapor form would be introduced to the bacteria in a glass tube. Then, optical activity can be measured to determine how much of the BTEX component was degraded. This data could be used to compare the degradation rate in literature with the degradation rate of the genetically modified co-culture.
The rate in which the components of BTEX diffuse through the fiberglass shell of the reactor is concerning. The regulations of volatile carbon emissions must be met during the use of BTEXterminator. Thus, the understanding of whether the residence time of one hour is
acceptable to assume negligible emissions must be known in order to determine the best material
for the reactor. One way of measuring this in the lab is by placing the vapor of a component in a
vessel of the material to be tested to see whether the one-hour residence time is acceptable in
meeting the emissions regulations. This will be determined by measuring how much of the
component is left in the vessel after one hour. Then, the diffusion rate of the components of
BTEX at the typical levels of produced water can also be tested. That way, if the tank can handle
a high concentration of BTEX in that residence time, it can be assumed that the tank can handle
the typical levels of BTEX in produced water. This analysis will determine the reactor material.
Design Alternatives (Juliet)
Hollow Fiber Membrane bioreactor
Membrane bioreactor (MBR) technology has seen widespread use due to a few advantages it
holds over conventional treatment processes. MBR systems have a smaller footprint, and they
provide better effluent quality, and better process control
16. Initially, MBR systems were
designed using cross flow micro- or ultrafiltration membrane systems, which limited their use
due to the high energy demand. In the late 80s, a new approach was developed where the
membrane modules were submerged in a tank, aeration was used to induce cross-flow and the
effluent was removed using a vacuum as shown in Figure 11
16. C&C can investigate the use of a
hollow fiber MBR in its process as an alternative to the fibrous bed bioreactor utilized in the base
case. Even though it has several advantages, further research would be needed to ensure that a
hollow fiber MBR is a viable alternative to house the P. putida co-culture.
Figure 11. MBR Module.15
Figure 12 shows the patented PURON module, a system C&C could implement in the
process
15. In this PURON module, the hollow fibers are sealed at the upper end and allowed to
float freely since they are only fixed at the bottom. An MBR module with hollow fibers that are
fixed both at the top and the bottom usually experiences a build-up of fibrous materials that can
clog the upper ends of the fibers
15. As shown in Figure 12, bacteria, and any solids in the
produced water, remain on the outside of the hollow fiber membrane while the treated water is
pulled inside the hollow membrane with a vacuum. A high flow-rate is achieved with the
outside-to-inside flow pattern as shown in Figure 12
15.
Figure 12. MBR Demo: MBR module and cassette; the cassette as shown on the left would be installed on the sk id.15
A big disadvantage of using hollow fiber MBR systems is poor cell observation and harvesting
17. The P. putida strains in this MBR would be freely suspended, while the cells growing in the fibrous bed reactor would be immobilized. Immobilized cell cultures hold the following advantages over suspension cultures as outlined by Shuler and Kargi
17:
1. Immobilization allows for high cell concentrations.
2. Immobilization allows the cells to be reused, thus eliminating the potentially costly process of cell recovery.
3. When dilution rates are high, immobilization eliminates cell washout problems.
4. Immobilization yields high volumetric productivities due to the combination of high cell concentrations and high flow rates.
5. Immobilization may result in higher product yields and rates since it creates favorable micro environmental conditions for the cells such as cell-cell contact, nutrient-product gradients and pH gradients.
6. Immobilization also provides cells with protection against shear damage.
If C&C were to truly consider the use of a hollow fiber MBR as an alternative design for water treatment, there are several design requirements that need to be addressed during the research phase. When designing a MBR system, there needs to be a way to control or reduce the formation of biological foam in the reactor. Filamentous bacteria that float can be trapped inside the MBR, causing biological foaming which “results in undesired loss of biomass and increased membrane fouling rates”
15. These filamentous bacteria may be found in the produced water that enters C&C’s water treatment process. A solution to this problem could be installing a front-side ultrafiltration and UV disinfection system as shown in Figure 15. The front-side ultrafiltration and UV disinfection system would ensure that any bacteria that is present in the feed produced water does not enter the hollow fiber MBR. Additionally, the produced water may contain high enough mineral or metal concentrations that will contribute to membrane fouling. Membrane fouling will increase C&C’s operation and maintenance costs in the form of additional chemical cleans necessary for optimum membrane operation. C&C will need to select membrane materials that are reliable and energy efficient.
Figure 13. Hollow Fiber Reactor PD: Overall Process and production diagram using a hollow fiber membrane bioreactor.
Figure 13 shows the alternative process diagram where C&C uses a hollow fiber MBR.
The hollow fiber MBR is set up to keep the P. putida cells outside and the treated water inside the fibers as shown in Figure 12. If a hollow fiber MBR is used, C&C will eliminate the need for an ultrafiltration membrane unit after the bioreactor. C&C would just need to install a UV
disinfection to kill any bacteria that manages to escape with the effluent.
Figure 14. Fibrous Reactor PD: Overall process and production diagram using a fibrous bed bioreactor.
